Favored Sites for HIV cDNA Integration in the Human Genome Abstract In order to replicate, a retrovirus must integrate a DNA copy of its RNA genome into a chromosome of the host cell. The question of how retroviruses select genomic sites for DNA integration has long interested retrovirologists, because studies in this area provide a unique window on replication mechanism and inform design of potential antiviral therapy. We have investigated integration targeting in detail, taking advantage of new high-throughput sequencing and bioinformatic methods. Studies of integration targeting have emphasized the importance of a new cellular factor LEDGF/p75, which has in turn suggested new routes to designing inhibitors. Highlights of published data from the previous funding period include the findings that 1) HIV favors integration in active transcription units, 2) other retroviruses, in contrast, show different favored genomic integration sites, 3) the cellular protein PSIP1/LEDGF/p75, which binds tightly to HIV integrase (IN), helps guide HIV integration into transcription units, 4) swapping the MLV IN coding region into HIV causes the HIVmIN chimera to integrate with the specificity of MLV, 5) applying the ultra-high throughput pyrosequencing method has allowed 40,761 unique HIV integration sites to be determined, revealing that integration takes place on histones in chromosomal DNA and that histone post-translational modifications are important determinants of HIV integration site selection in vivo. Our Specific Aims for the renewal are as follows. In Aim 1, we will study integration site selection by tethering of integration complexes to cellular factors. We will extend our studies of the HIV IN binding protein PSIP1/LEDGF/p75 to investigate the mechanisms by which this factor promotes integration efficiency and guides integration targeting. We will also use genomic methods to identify additional candidate factors important in HIV integration, and biochemical methods to study their mechanisms. In Aim 2, we will investigate the influence of chromatin structure on integration, with a focus on extending the observations from pyrosequencing data mentioned above. In Aim 3, we will investigate the possible role of cell cycle progression in integration targeting. In the previous funding cycle, our data sets and methods were used extensively by other laboratories--in the renewal, we aim to contribute an even richer set of resources to the field. PUBLIC HEALTH RELEVANCE: The attached grant application is designed to improve our understanding of HIV DNA integration. The integration system is a target for antiviral agents, and the proposed experiments should provide information directly useful for designing inhibitors.